The long term goals of this project are to understand the molecular mechanisms of cilia biogenesis. Towards that end two Tetrahymena thermophila mutants with temperature-sensitive defects in both cilia biogenesis and some aspect of the cell cycle will be analyzed. When deciliated one of the mutants regenerates paralyzed cilia. When incubated in growth medium it fails to complete cytokinesis. The molecular basis for cilia paralysis will be determined by comparing motile and paralyzed cilia by 2-D polyacrylamide gel electrophoresis and by electron microscopy. Wild-type and mutant cilia will be compared by 2-D polyacrylamide gel electrophoresis to identify the gene product encoded by the defective gene. The cellular location of the gene product in growing cells will be determined. The other mutant fails to initiate cilia biogenesis following deciliation and arrests in late micronuclear G2 when incubated in growth medium. To determine whether the defective gene in that mutant encodes a kinase required for cilia biogenesis and for cell cycle progression, the phosphorylation state of basal bodies at permissive and restrictive temperatures before and following deciliation will be determined. A temperature-sensitive kinase activity will be identified in extracts of mutant cells. To identify additional genes involved in cilia biogenesis and/or the cell cycle, extragenic suppressors of the two mutations will be isolated. Cilia and flagella are nearly ubiquitous in eukaryotes and function in the human reproductive and respiratory tracts, and dysfunction of the organelles has been implicated in several disease states. An understanding of the molecular mechanisms of cilia and flagella assembly and the way assembly is coordinated with the cell cycle will contribute to an understanding of the mechanisms by which assembly of all microtubules is regulated. This is important because abnormalities of the cytoskeleton are often observed in neoplastic cells.